Our proposal is focused on how CRAF signaling in vascular cells promotes endothelial cell survival and resistance to different forms of cellular stresses present within the tumor microenvironment. In addition to activating the kinase-dependent RAS-RAF-MEK-ERK pathway, CRAF phosphorylation on Serine 338 (pS338) represents an additional target that could be suppressed to improve the effects of cancer therapy on tumor blood vessels. Mechanistically, we established that CRAF pS338 functions as a molecular scaffold to recruit effectors that drive a variety of signaling pathways, such as the cell cycle kinase PLK1 and the DNA repair enzyme CHK2. Activation of these pathways can be blocked by expressing a non-phosphorylatable CRAF S338A mutant or by treatment with 3G8, an allosteric inhibitor we developed that not only blocks the kinase-dependent activation of MEK/ERK, but also holds CRAF in an inactive conformation to suppress pS338. Based on these studies, we propose that a CRAF interactome represents a significant factor in endothelial cell resistance to genotoxic stress. In addition to PLK1 and CHK2, computational modeling was used to identify additional kinases predicted to couple with and be activated by the kinase-dead form of CRAF, and a mass spectrometry screen was performed to identify proteins that favor association with CRAF when phosphorylated on S338. In the following Specific Aims, we will test the hypothesis that disabling CRAF's ability to couple with and activate these effectors will enhance the damaging effects of stress and sensitize tumor-associated blood vessels to therapy. Aim 1: Evaluate functions of kinase-dead CRAF as a scaffold and kinase activator in EC Aim 2: Test the ability of RAF blockade to potentiate activity of cell cycle inhibitors, DNA damaging agents, and anti-angiogenics Aim 3: Target non-canonical CRAF functions to disrupt tumor angiogenesis in vivo If successful, our studies should generate broad interest. From a clinical perspective, strategies to target CRAF pS338 could graduate to clinical testing for a broad population of cancer patients with the possibility of enhancing anti-tumor activity and lowering the chemo/radiation dose required for efficacy. At the basic science level, we believe we have identified a new kinase-independent function for a kinase, and that what we learn about this new scaffolding function of CRAF could be applied to other kinases.